IDENTIFICATION AND CHARACTERIZATION OF ESCHERICHIA-COLI DNA HELICASE-II MUTANTS THAT EXHIBIT INCREASED UNWINDING EFFICIENCY

Using a combination of both ethyl methanesulfonate and site-directed m utagenesis, we have identified a region in DNA helicase II (UvrD) from Escherichia coli that is required for biological function but lies ou tside of any of the seven conserved motifs (T. C. Hodgman, Nature 333: 22-23, 1988) associated with the superfamily of proteins of which it i s a member. Located between amino acids 403 and 409, alterations in th e amino acid sequence DDAAFER lead to both temperature-sensitive and d ominant uvrD mutations. The uvrD300 (A406T) and uvrD301 (A406V) allele s produce UV sensitivity at 44 degrees C but do not affect sensitivity to methyl methane-sulfonate (MMS). In contrast, the uvrD303 mutation (D403AD404A) causes increased sensitivity to both UV and MMS and is do minant to uvrD(+) when present at six to eight copies per cell. Severa l of the alleles demonstrated a strong antimutator phenotype. In addit ion, conjugal recombination is reduced 10-fold in uvrD303 strains. Of all of the amino acid substitutions tested, only an alanine-to-serine change at position 406 (uvrD302) was neutral. To determine the biochem ical basis for the observed phenotypes, we overexpressed and purified the UvrD303 protein from a uvrD Delta 294 deletion background and char acterized its enzymatic activities. The highly unusual UvrD303 protein exhibits a higher specific activity for ATP hydrolysis than the wild- type control, while its K-m for ATP binding remains unchanged. More im portantly, the UvrD303 protein unwinds partial duplex DNA up to 10 tim es more efficiently than wild-type UvrD. The DNA binding affinities of the two proteins appear comparable. Based on these results, we propos e that the region located between amino acids 403 and 409 serves to re gulate the unwinding activity of DNA helicase II to provide the proper balance between speed and overall effectiveness in the various DNA re pair systems in which the protein participates.